Microbiological Profile of Kashkaval Cheese During Production Season
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Mirela1 Department Anamaria of Food JIMBOREAN Engineering,, Dorin Faculty ŢIBULCĂ of Food*, DeliaScience MICHIU and Technology, University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur Street, Cluj-Napoca, România *corresponding author: [email protected]
ISSN-L 2344-2344; Print ISSN 2344-2344; Electronic ISSN 2344-5300 DOI:Bulletin 10.15835/buasvmcn-fst: UASVM Food Science and 2019.0042 Technology 77(1)/2020
Abstract
Although the scalded cheeses are not an ideal substrate for microorganisms, these are a suitable habitat for starter cultures and indigenous microbiota, the latter being able to survive during processing. The aim of this study indicators:was to highlight determination the qualitative of coliform and hygienic bacteria aspects, E. ofcoli kashkavals as a finished products. The microbiologicalStafylococcus assays aureusaimed thecoagulase-positive evaluation of sanitary-hygienic quality in kashkaval cheeses obtained during one year, by the following , monitoring of pathogenic microorganisms: between 102-103 and total number of micromycetes. Sixty samples2 fromcfu/g. five 8.3% units of cheese which samples process had the valuesmilk into of scaldedE. coli ranging cheeses, from were 11 collected. to 100 cfu/g 10% and from the theremaining analyzed samples cheese hadsamples, lower had values values. No ofsample coliform registered counts cfu/g, while the remaining samples had values less than 10 whileover 1,000 the remaining cfu/g, which samples is the were upper below marginal 1000 limitcfu/ g.for coagulase-positive staphylococci in cheeses, according to the Reg. EC 1881/2006. 18.3% of cheese samples had values of total number of micromycetes over 1000 cfu/g, Keywords:
coliform bacteria, micromycetes, scaled cheese
Introduction The main feature of the technological process general microbiological quality is very difficult, sometimesColiform impossible bacteria is a to sanitary perform, microbiological due to the of Kashkaval cheese is to obtain the curd, which, indicatorintervention with of microflora a special used meaning. in their production. Coliform after ripening and cutting (into slices of 3-5 mm bacteria constitutes a dangerous group among thickness and 3-6 cm width), is scalded at 74-80° andC (Costin a large et part al., 2003). of it is still practiced on a relatively Cheese making is a major industry worldwide, microorganisms,The occurrence that of infectpsychrotrophic cheeses bacteria through various sources (Giammanco et al., 2011). small scale, which accounts for the rich diversity may lead to excessive accumulation of free fatty cheesesof available have cheeses a very (Fox microbiologically et al., 2004). complex acidsin cheeses and rancid causes aromas defects in likebrined lipolysis, cheese which. This Bărzoi and Apostu (2002), showed that system. Considering this, the assessment of their is the frequent result of milk contamination 92 et al.
JIMBOREAN with psychotrophic bacteria that produces Starting with the number of positive test heat-resistantYeast and molds lipolytic can enzymes.appear on (Bintsisthe cheeses and tubes,In order the most to establish probable the number number of of coliform E. coli Papademas, 2002). bacteria (NCP) per ml (g) sample was calculated. , during the manufacturing process, from the air, accordingSR EN to ISO SR ISO6888-1-2/A1/2005 16649-2 / 2007, thestandard most machinery, installations, containers, packings, or establishesprobable number the horizontal technique methodwas used. for counting limitduring for storage cheeses and is 1000 packaging. yeasts According and molds/g to the of colonies of coagulase-positive staphylococci on Ministry’s Order (M.S.) from 1998, the maximum not provide any informations for this indicator. In product. The new Regulation EC 1881/2006, does Baird-Parker medium after aerobic incubation during (35-37°C),The coagulase-positive from products intended staphylococci for human counts or this study, we analised the microbiological quality peranimal ml orconsumption. g of sample (Rotarwas made et al., from 2015). the number determineand sanitary the status number of of Kashkaval coliform bacteria cheese and E. of typical and/or atypical colonies obtained on the coli,a year coagulase-positive of production, the staphylococci main purpose and beingthe total to . by coagulase-positive assay. Yeasts and molds are selective medium and subsequently confirmed numberMaterials of micromycetes. and Methods (Prates et al., 2017) selective medium. SR ISO 7954/2001 standard Sampling providesmicroorganisms the counting which at of 25°C yeasts form and colonies molds on
result was expressed as a number between 1.0 and In this study, the manufacturing process of 9.9according multiplied to the by counting10 technique at 25° C. The curd involved the following steps: cow’s milk Statistical analysisX reception, cleaning and standardization of milk, The data were analyzed, where x by is theTwo-way power ANOVA of 10. milk pasteurization (62-65°C for 20-30 minutes) and cooling (35-38°C), preparation for coagulation different time points. which consisted in adding calcium chloride (10 - test, to compare the microbial levels between 20 g/100 liters ofLactococcus milk, increasing lactis the subsp coagulation. lactis Results and discussions Streptococcusability of the milkthermophilus ) and selectedand Lactobacillus cultures of The statistical analysis of microorganisms caseilactic bacteria ( , then ).made The coagulationand the resulting of milk curd was wasachieved ripened at 32- at in kashkaval samples is ± s presented in table 1. 35°C, for 30–40 minutes. Processing of curd was Statistically, in relationX to X the total number of 5.0. The ripened curd was cut into slices having andsamples, 460.0 the cfu/g. average The ( average) of of coliform E.coli bacteriawas log 18-26°C for 6-10 hours until a pH between 4.8- was log. 1.153 ± 0.09592 with values between 0.0 cfu/g while for coagulase-positive staphylococci ina thicknessbrine with between 10–12% salt. 0.3 -The 0.5 scalded cm and pasta width was of 0.2172 ± 0.06007 for values ranging from 0 to 30.0 3-6 cm and was scalded at 72-74°C for 50–60 s were between 0.0 and 130.0 cfu/g. Regarding was recorded 0.3723 ± 0.06958. The values then ripened. Storage was done at 4-6°C and φ = for 0.0 and 2.1 x 104 cfu/g. 85–90%. (Costin et al., 2003). micromycetes,Coliform bacteria the average was log. 2.349 ± 0.1042 per Sixtyseason. samples For the of kashkaval (semi-hard texture) samplesfrom five were units transported were analysed, under fifteen refrigerated samples bacteria /g in cheese was performed. In the conditions and were microbiologicalstored in a refrigerator analysis, allat In this study, a seasonal analysis of coliform between 0-10 cfu/g and 26.7% between 11-102 Microbiological analysis autumn, 73.3% of samples had coliform counts 2-4ºCTotal and analyzedcoliform within counts 24 hours.was performed of 0-10 cfu/g and 20% ranging from 11 to 102 according to the SR ISO 4831/2009 standard. cfu/g.cfu/g while in the winter, 80% with a mean value
Bulletin UASVM Food Science and Technology 77 (1) / 2020 93
Microbiological Profile of Kashkaval Cheese During Production Season Table 1. during production season The statistical analysis of microorganisms in pressed kashkaval Dalia Log. of Standard Minimum Maximum Log. of Season Samples ppm ppm X s error of theX mean, value, value, Winter 15 0.00 50.00 Mean,0.586 0.148 Coliform Spring 15 0.00 100.00 1.140 0.144 bacteria Summer 15 8.00 460.00 1.989 0.126 Autumn 15 0.00 70.00 0.897 0.137 Winter 15 0.00 0.00 0.0 - Spring 15 0.00 0.00 0.0 - E.coli Summer 15 0.00 30.00 0.802 0.154 Autumn 15 0.00 10.00 0.067 - Winter 15 0.00 20.00 0.087 - Coagulase- Spring 15 0.00 130.0 0.563 - positive staphylococci Summer 15 0.00 20.00 0.649 0.131 Autumn 15 0.00 9.00 0.191 0.091 Winter 15 0.00 520.00 1.671 0.194 Spring 15 10.00 820.00 2.209 0.149 Micromycetes Summer 15 20.00 21000 3.151 0.174 Autumn 15 40.00 1100 2.365 0.111
Sources of coliforms in cheese products can 2 vary depending on the product. Due to the nature In the spring, 40% of samples registered 0-10 cfu/g and 60%, 11-10 cfu/g coliforms counts, in not unexpected as coliforms are common in raw 11-10relation2 cfu/g to the and summer, 40% of where, samples 6.7% with of coliform samples of raw milk cheeses, the presence of coliforms is countswere in ranging the range from of 100-102 to cfu/g,103 cfu/g. 53.3% between Detection of coliform bacteria is not a milk. However, in pasteurized cheese products, coliforms present in raw milk should have been eliminated by pasteurization, implying that any poorcompulsory hygiene test, conditions but their and presence a lower in cheesesactivity coliforms present in the finished product resulted involves inadequate pasteurization of raw milk, fromEscherichia post-processing coli contamination (Martin et al. coliform count is considered a good predictor 2016).Seasonal analysis of E. coli counts/g in of lactichygienic microflora and sanitary in manufacturing. practices during The totalfood
kashkaval showed the following results: in the production (Costello et al. 2014). autumn, 93.3% of samples had values in the range Wolfe et al. (2014) reported that cheese of 0-5 cfu/g, and 6.7% had values between 6-10 product characteristics, including moisture ofcfu/g, samples while registered in the winter, E. coli 100% counts of betweensamples had0-5 andcontent, roles pH, for salt coliforms content, and ripening other microorganisms conditions, age values in the range of 0-5 cfu/g. In spring, 100% of product, and culture, all influence potential levels Even though coliforms are considered 6-10cfu/g cfu/g in relation and 33.3% with betweenthe summer, 11-10 where2 cfu/g. 46.7% in the final product. ranged between 0-5 cfu/g, 20% ranged between L. monocytogenes and E. coli O157: H17 thermolabile and do not survive pasteurization, Pasteurization of milk destroys pathogenic Trmčić et al. (2016), found that 21% of pasteurized Survivalbacteria, butdepends on many factors: initial level of milk cheeses tested positive for coliforms (>10 can survive and contaminate the finished product. cfu/g). Bulletin UASVM Food Science and Technology 77 (1) / 2020 94 et al.
JIMBOREAN Table 2. The statistical differences regarding the microorganisms in samples during
kashkaval production season (Two-way ANOVA)Difference between Parameter Comparison % of total variation P value means of differences A - W 7.82 0.0996 Significancens A - Sp 5.06 0.1375 ns 0.3113 ± 0.2021 Coliform bacteria. A - Su 55.06 *** -0.2427 ± 0.1987 log. cfu/g W - Sp 20.42 0.0108 * -1.092 ± 0.1864 P<0.0001 W - Su 64.94 *** -0.5540 ± 0.2067 Sp - Su 41.28 0.0002 *** -1.403 ± 0.1949 P<0.0001 A - W - - - - -0.8493 ± 0.1914 A - Sp - - - - A - Su 40.78 0.0009 *** E. coli. log. cfu/g W - Sp - - - - -0.7353 ± 0.1675 W - Su 40.78 0.0009 *** Sp - Su 40.78 0.0009 *** -0.7353 ± 0.1675 A - W 2.40 0.4519 ns -0.7353 ± 0.1675 A - Sp 10.88 0.0904 ns Coagulase- 0.1040 ± 0.1253 positive A - Su 22.77 0.0195 * -0.3727 ± 0.2016 staphyloccoc. W - Sp 16.89 0.0059 ** -0.4580 ± 0.1594 log. cfu/g W - Su 31.32 0.0007 *** -0.4767 ± 0.1999 Sp - Su 0.52 0.6774 ns -0.5620 ± 0.1573 A - W 25.61 0.0066 ** -0.08533 ± 0.2228 A - Sp 2.45 0.2403 ns 0.6947 ± 0.2238 Micromycetes. A - Su 34.00 0.0004 *** 0.1560 ± 0.1862 log. cfu/g W - Sp 14.75 0.0548 ns -0.7853 ± 0.2068 W - Su 53.50 *** -0.5387 ± 0.2448 Sp - Su 37.56 0.0001 *** -1.480 ± 0.2608 P<0.0001 Legend: A/ autumn; W/ winter; Sp/ spring; Su/-0.9413 summer. ± 0.2293 ns – not significant (p > 0.05); * – significant (0.01 < p < 0.05); ** – very significant (0.001 < p < 0.01); *** – extremely significant (p < 0.001). E.coli. In 44% of the samples. E. coli exceeded 103 CFU g-1 and in 8% of the samples its presence was contamination, pasteurization temperature, acid 2 and 103 CFU g-1. conditions.and salt tolerance of microorganisms, competitive microflora,Pasteurization cheese composition is able to anddestroy manufacturing essential detected in quantities between 10 playedThe a resultsmajor role of a to study the contamination released by Kwenda, of the It should be noted that the inactivation level cheddar2015, showed cheese thatwith poorE. coli hygiene and coliform practices bacteria had ofmicroflora, microorganisms enzymes depends and pathogens on the amount in milk. of
as well as other factors like pasteurization microorganisms, growth phase and other factors ofefficiencies. E. coli (Ciprovica and Mikelson. 2011). showedBaranceli that up et al.to (2014),93% of studied samples the commercially percentages Giammanco et al. (2011), analyzing the available at counts sales inpoints packaged were Minasdetected cheeses with fecal and appearedmicrobiological not to quality be satisfactory of Pecorino according Siciliano to coliforms above the tolerance limit established Europeancheese, found Regulation 27 of the 2005/2073/EC. 50 samples (54%) This thatwas by legislation. The higher percentages were mainly due to high levels of glucuronidase-positive
indicative of lower hygienic conditions in the final Bulletin UASVM Food Science and Technology 77 (1) / 2020 95
Microbiological Profile of Kashkaval Cheese During Production Season
toxicity. Many studies showed that the foodstuffs steps of cheese manufacture, including molding, contaminatedwith toxic effects with like thecarcinogenic, toxigenic developmentalmicromycetes packaging. have presented a serious hazard of the „hidden Istrati et al. (2006), in one of their studies, samplehave analyzed with E. coli11 Rucvalues and below Dalia 10 cheese cfu/g. samples, Regarding the statistical analysis of the sold Somein the of county the microorganisms of Bacău and have that found grow ina single dairy mycotoxins” (Ostry et al., 2004). E. coli products are able to produce undesirable reactions that deteriorate the organoleptic characteristics logarithmic mean values of coliforms, and micromycetes, from the kashkaval samples during the production season, have been observed of cheese,Coagulase-positive while other can staphylococci potentially cause food- extremely significant differences for (A-Su), borne diseases (Lu et al.. 2013).3 cfu/g which is the (W-Su) and (Sp-Su), while coagulase-positive staphyloccoc have been reported very significant No sample had values > 10 differences for (W-Sp). 1881/2006.upper marginal limit (M) for coagulase-positive Insignificant levels of (A-W), (A-Sp) production staphylococciThe presence in of cheeses, these microorganisms according to Reg. in dairy EC were induced by coliformmicromycetes bacteria, .(A-W), (A-Sp) and (Sp-Su) by coagulase-positive Staphyloccoc and Conclusions(A-Sp), (W-Sp) by products depends by factors such as the quality According to the results of this study the of raw material, the respect of good practices of productionMicromycetes and storage conditions (Ledenbach and TheMarshall. determinations 2009). of micromycetes highest coliform bacteria values in kashkaval counts/g during the four seasons showed the cheeses were detected in the summer, ranging fromAnalyzing 8.0 to 460.0 the seasonal cfu/g, and variations the lowest of E.coli in . the in winter, between 0.0 and 50.0 cfu/g. following dates: in the autumn, 26.7% of the 501-1000samples had cfu/g values and ranging6.7% were from over 0 to 1000 100 cfu/g.cfu/g, andthe winter 30.0 cfu/g. and spring According was absent,to the Ministry while the of highest Health 53.3% between 101-500 cfu/g, 13.3% between values were in the summer, ranging between 0.0 cfu/gIn the and winter, one sample 66.7% between of samples 501-1000 had cfu/g. values In molds/gRegulations product. from 1998,The new up toregulations 10 coliforms/ do not g between 0-100 cfu/g, 26.7% between 101-500 provideproduct any are information allowed, maximum for these 1000 indicators. yeasts and The highest level of coagulase-positive andthe spring,26.7% 33.3%between of 501-1000samples had cfu/g values while ranging in the staphylococci group of bacteria were detected in from 0 to 100 cfu/g, 40% between 101-500 cfu/g summer, 6.7% of samples had a range of 0-100 whilethe spring, the highest between Micromycetes 0.0 and 130.0 counts cfu/g were and in the 18.3%cfu/g, 26.7%of all collected were between and analyzed 501-1000 samples cfu/g andhad lowest in the autumn, between 0.0 and 9.0 cfu/g 66.7% with micromycetes counts over 1000 cfu/g, in the winter between 0.0 and 520.0 cfu/g. Most problems raised by the contamination summer,The 20.0microrganisms - 21000.0 cfu/g that and are the indicators lowest were of values > 1000 cfu/g. to their shelf life. These microorganisms have a bacteria, E.coli and micromycetes were present in strongof kashkavals proteolytic with and yeasts lipolytic and moldsactivity. have In order related to the sanitary-hygienic quality, such as coliform the summer. kashkavalThe results samples of atthis relatively study highlighthigh levels that during the present no risks for human health and to extend lesstheir than shelf-life, 1000/g the of kashkavalproduct must not contain such microorganisms, or their number should be high temperatures during the summer influenced (Bărzoi and Apostu. necessarythe microbial to improve load. Due hygienic to the practices risks that during these are2002). important factors with a potentially negative microorganisms represent to public health, it is Ostry et al., 2004, reported that micromycetes the manufacturing process. the warm season, both at the reception and during effect on human health, especially if are toxigenic, Bulletin UASVM Food Science and Technology 77 (1) / 2020 96 et al.
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